Preparation Of Bound Gossypol And Its Acute And Chronic Toxicity Experiment

In this paper ethanol solution of Hydrochloric acid was used for the extraction of gossypol from the cottonseed meal with the preliminary purification of acetone and ether.Cottonseed protein was extracted from phenol removal cottonseed meal with lye.It obtained a group of optimized parameters in the process.After preliminary purification and protein synthesis reaction with gossypol,Binding gossypol was obtained;and then animal experiments of binding gossypol was prepared for acute toxicity and sub-chronic toxicity tests,as well as its digestion and tissue distribution study,the results are as follows:The method we used was from our own laboratory.We used Hydrochloric acid-ethanol solution for the extraction of crude gossypol,and then 80%of acetone and diethyl ether was used for preliminary purification.By utraviolet spectrophotometry the purity of gossypol was 61%.By using the UV scanning analysis and IR scanning analysis,it showed a typical characteristic absorption peaks and the structural characteristics of gossypol.This experiment collect protein from the dephenolization cottonseed meal,and design the response surface methodology by the Box-Behnken to find the optimum process conditions for the alkali extraction of cottonseed protein:extraction temperature 61?,ratio of material to liquid 1:20,extraction time 90min and pH 10.7.The highest content of cottonseed protein in the experiment reached up to 91%,and the color was similar to milkiness.What's more,the content of the free gossypol and the bound gossypol is respectively 0.0023%.0.00275%,it'll increase the safety and utilization value of the cottonseed protein efficiency.The purified gossypol and protein were used for the Preparation of bound gossypol.Ethanol solution with 2mg/mL of gossypol and 200 mg/mL of protein suspension were mixed in a volume ratio of 1:1,and then synthesize with the pH value of 7.5 and the temperature of 40 ?.a small amount of gossypol could be not participated in the reaction,so it was needed to remove the free gossypol.by single factor experiment to determine the optimum conditions,the ultrasonic power was 80W,liquid to solid ratio was 25mL/g,time was 50min,temperature was 50?,By determination of phloroglucinol results:the content of total gossypol was 0.76%,the content of free gossypol was 0.0014%.The content of free gossypol was far below the national standard of 0.02%.Acute toxicity and subchronic toxicity experiments of bound gossypol to provide theoretical basis for the rational utilization of the cottonseed protein.In this experiment,take white mice as the research object to test the toxicity by pouring into stomach.The results show that the bound gossypol's acute oral toxicity is low which LD50>10g/(kg·d).And three doses of bound gossypol(lmg/10g?5mg/10g?10mg/10g)for successive administration 3d shows that the appetite and weight of every experimental group all increase slowly,what's more,the lesion phenomenon of the mice extirpated for checking isn't observed,there is no significant differences with every experimental group for the organ coefficient.However,in the high dose group,the toxic and side effects of the ALT in liver and the LDH in testis is significant,so that we can conjecture that the bound gossypol can be digested and resolved into free gossypol become toxicity.Investigate the bound gossypol digesting and resolving in the stomach and Study the digesting of bound gossypol in sim?Lating gastric juice.Stock diet was given with 10mg/lOg gossypol to each group by using stomach tube every day for 30 days,It was not detected in blood samples of gossypol by HPLC.And then we Use sim?Lated artificial gastric juices A,B on the binding of gossypol digestion study.The effect of pH on the gossypol content can be measured with the ?Ltraviolet spectrophotometer at different time.The res?Lts show that the effect of gossypol digesting become significant as the pH 1.5,as pH increase,the gossypol degradation decrease,and the gastric juice A is stronger than B as the pH 1.5,instead,the gastric juice A is weaker than B,the reason can be that pepsin has the most effect of digestion at pH 1-2.As the pH is higher,it will be less efficient.What5s more,the effect on the sim?Lating gastric juice digestion of bound gossypol will reduce after 240min,which indicates that the free gossypol may bond with the amino acid and protein.